Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

Download Full-text

Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

Scientific Reports ◽

10.1038/s41598-019-49474-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Dorota M. Krzyżanowska ◽

Anna Supernat ◽

Tomasz Maciąg ◽

Marta Matuszewska ◽

Sylwia Jafra

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Reference Genes ◽

Significance Threshold ◽

Expression Studies ◽

Reverse Transcription Quantitative Pcr ◽

Gene Expression Studies ◽

The Stability ◽

Selection Of

Abstract Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

Download Full-text

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages

Frontiers in Genetics ◽

10.3389/fgene.2018.00269 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 6

Author(s):

Lifeng Zhou ◽

Fengmao Chen ◽

Jianren Ye ◽

Hongyang Pan

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Developmental Stages ◽

Bursaphelenchus Mucronatus ◽

Qpcr Analysis ◽

Selection Of

Download Full-text

Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes

Genes ◽

10.3390/genes11010113 ◽

2020 ◽

Vol 11 (1) ◽

pp. 113 ◽

Cited By ~ 4

Author(s):

Mengyao Li ◽

Fangjie Xie ◽

Qi He ◽

Jie Li ◽

Jiali Liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Expression Patterns ◽

Regulatory Mechanisms ◽

Future Research ◽

Transcriptome Data ◽

Accurate Analysis ◽

Report Analysis ◽

The Stability ◽

Selection Of

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

Download Full-text

Selection of reliable reference genes for gene expression analysis in seeds at different developmental stages and across various tissues in Paeonia ostii

Molecular Biology Reports ◽

10.1007/s11033-019-05036-7 ◽

2019 ◽

Vol 46 (6) ◽

pp. 6003-6011 ◽

Cited By ~ 3

Author(s):

Chaoqiong Li ◽

Lizong Hu ◽

Xueqin Wang ◽

Hongzhan Liu ◽

Huihui Tian ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Developmental Stages ◽

Selection Of

Download Full-text

Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance

Rice ◽

10.1186/s12284-016-0104-7 ◽

2016 ◽

Vol 9 (1) ◽

Cited By ~ 17

Author(s):

Isaiah M. Pabuayon ◽

Naoki Yamamoto ◽

Jennylyn L. Trinidad ◽

Toshisangba Longkumer ◽

Manish L. Raorane ◽

...

Keyword(s):

Gene Expression ◽

Drought Tolerance ◽

Reference Genes ◽

Developmental Stages ◽

Gene Expression Analyses ◽

Different Tissues

Download Full-text

Evaluation of putative reference genes for quantitative real-time PCR normalization inLilium regaleduring development and under stress

PeerJ ◽

10.7717/peerj.1837 ◽

2016 ◽

Vol 4 ◽

pp. e1837 ◽

Cited By ~ 15

Author(s):

Qiang Liu ◽

Chi Wei ◽

Ming-Fang Zhang ◽

Gui-Xia Jia

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Real Time ◽

Quantitative Pcr ◽

Biotic Stress ◽

Reference Genes ◽

Developmental Stages ◽

Real Time Quantitative Pcr ◽

The Stability ◽

Experimental Treatments

Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression,Lilium, and particularlyL. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely18S rRNA,ACT,BHLH,CLA,CYP,EF1,GAPDH,SANDandTIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms,BHLHwas superior to the other candidates when all the experimental treatments were analyzed together;CLAandEF1were also recommended by two of the three algorithms. As for specific conditions,EF1under various developmental stages,SANDunder biotic stress,CYP/GAPDHunder drought stress, andTIP41under salinity stress were generally considered suitable. All the algorithms agreed on the stability ofSANDandGAPDHunder cold stress, while onlyCYPwas selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level ofLrLOXin leaves inoculated withB. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding ofLilium.

Download Full-text

Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum

PeerJ ◽

10.7717/peerj.7133 ◽

2019 ◽

Vol 7 ◽

pp. e7133 ◽

Cited By ~ 4

Author(s):

Wen Zhou ◽

Shiqiang Wang ◽

Lei Yang ◽

Yan Sun ◽

Qian Zhang ◽

...

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Reference Genes ◽

Hypericum Perforatum ◽

Developmental Stages ◽

Pcr Analysis ◽

Potential Candidate ◽

Qrt Pcr ◽

Expression Studies ◽

Different Tissues

Hypericum perforatum L. is a widely known medicinal herb used mostly as a remedy for depression because it contains high levels of naphthodianthrones, phloroglucinols, alkaloids, and some other secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is essential for the interpretation of qRT-PCR data. In this study, we investigated the expression of 14 candidate genes, including nine housekeeping genes (HKGs) (ACT2, ACT3, ACT7, CYP1, EF1-α, GAPDH, TUB-α, TUB-β, and UBC2) and five potential candidate genes (GSA, PKS1, PP2A, RPL13, and SAND). Three programs—GeNorm, NormFinder, and BestKeeper—were applied to evaluate the gene expression stability across four different plant tissues, four developmental stages and a set of abiotic stress and hormonal treatments. Integrating all of the algorithms and evaluations revealed that ACT2 and TUB-β were the most stable combination in different developmental stages samples and all of the experimental samples. ACT2, TUB-β, and EF1-α were identified as the three most applicable reference genes in different tissues and stress-treated samples. The majority of the conventional HKGs performed better than the potential reference genes. The obtained results will aid in improving the credibility of the standardization and quantification of transcription levels in future expression studies on H. perforatum.

Download Full-text

Selection of endogenous reference genes for gene expression analysis in Leishmania major developmental stages

Parasitology Research ◽

10.1007/s00436-007-0491-1 ◽

2007 ◽

Vol 101 (2) ◽

pp. 473-477 ◽

Cited By ~ 16

Author(s):

Meriam Ouakad ◽

Narges Bahi-Jaber ◽

Mehdi Chenik ◽

Koussay Dellagi ◽

Hechmi Louzir

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Leishmania Major ◽

Developmental Stages ◽

Endogenous Reference ◽

Selection Of ◽

Endogenous Reference Genes

Download Full-text

Selection of Suitable Reference Genes Based on Transcriptomic Data in Ginkgo biloba under Different Experimental Conditions

Forests ◽

10.3390/f11111217 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1217

Author(s):

Tingting Zhou ◽

Xiaoming Yang ◽

Fangfang Fu ◽

Guibin Wang ◽

Fuliang Cao

Keyword(s):

Gene Expression ◽

Ginkgo Biloba ◽

Reference Genes ◽

Developmental Stages ◽

Deciduous Tree ◽

Internal Reference ◽

Experimental Conditions ◽

Transcriptomic Data ◽

Suitable Reference ◽

Selection Of

Ginkgo biloba, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in G. biloba. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo.

Download Full-text

Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

10.1101/499186 ◽

2018 ◽

Author(s):

Zhezhi Wang ◽

Wen Zhou ◽

Lei Yang ◽

Yan Sun ◽

Qian Zhang ◽

...

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Reference Genes ◽

Hypericum Perforatum ◽

Developmental Stages ◽

Housekeeping Genes ◽

Potential Candidate ◽

Qrt Pcr ◽

Wide Range ◽

Different Tissues

Hypericum perforatum is a widely known medicinal herb used mostly as a remedy for depression because of its abundant secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is very necessary for the interpretation of qRT-PCR data. In this study, we investigated the expression of fourteen candidate genes, including nine housekeeping genes and five potential candidate genes. Additionally, the HpHYP1 gene, belonging to the PR-10 family associated with stress control, was used for validation of the candidate reference genes. Three programs were applied to evaluate the gene expression stability across four different plant tissues, three developmental stages and a set of abiotic stress and hormonal treatments. The candidate genes showed a wide range of Ct values in all samples, indicating that they are differentially expressed. Integrating all of the algorithms and evaluations, ACT2 and TUB-β were the most stable combination overall and for different developmental stages samples. Moreover, ACT2 and EF1-α were considered to be the two most applicable reference genes for different tissues and for stress samples. Majority of the conventional housekeeping genes exhibited better than the potential reference genes. The obtained results will contribute to improving credibility of standardization and quantification of transcription levels in future expression research of H. perforatum.

Download Full-text